This invention relates to the polypeptide ACSF (adenylate cyclase stimulating factor) and substances which antagonize the activity of ACSF in vivo. In particular, this invention relates to DNA encoding ACSF and methods for the use of such DNA to produce ACSF and its polypeptide antagonists, including amino acid sequence variants and antibodies directed against selected ACSF epitopes. This invention also relates to therapeutic compositions containing ACSF antagonists particularly for the treatment of hypercalcemia attendant upon various neoplasms.
A variety of cancers are clinically associated with nonmetastatic bone destruction and serum hypercalcemia (humoral hypercalcemia of malignancy, or HHM), most commonly breast, lung and skin carcinomas, but the phenomenon is by no means limited to these cancers. Soluble factor(s) released by the tumor cells which have been thought in the past to be responsible for HHM, include transforming growth factors, parathyroid hormone, prostaglandins, and other relatively uncharacterized factors. For an extensive review on this subject, see Mundy et al. , "New England Journal of Medicine" 310:1718 (1984). More recently, reports have appeared of substances partially purified from murine tumors, rat Leydig cell and human HHM tumors which stimulate the parathyroid hormone (PTH) receptor, exert adenylate cyclase activity, and are inhibited by the PTH antagonist Nle.sup.8,18,Tyr.sup.34 -bovine PTH (3-34) amide and which have a molecular weight in the range of 30-40 kD (Rodan et al., "J. Clin. Invest." 72:1511 [1983]; Strewler et al., "J. Clin. Invest.", 71:769 [1983]; Stewart et al. "Clin. Res." 32:410A [1984]; Merendino et al., "Science" 231:388 [1986]; Insogna et al. "Endocrinology" 120:2183 [1987]; Stewart et al. "J. Bone and Mineral Research" 1:267 [1986]; and Burtis et al. "Endocrinology" 118:1982 [1986 ) ). No amino acid sequence data for these factor(s) was disclosed by these authors, nor had the plurality of candidate factors been explained at a molecular level.
In work which, as of the filing date hereof, remains unpublished, Dr. T. J. Martin and colleagues have purified to homogeneity an ACSF from an HHM squamous carcinoma (BEN) cell extract and determined the amino terminal amino acid sequence of the factor:
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Uncertain residues are designated by asterisks.
Peptide analogues based on the first 17 residues from this sequence were synthesized: Ala-Val-Ser-Glu-His-Gln-Leu-Glu-His-Asn-Cys ([Glu.sup.8,Asn.sup.10,Cys.sup.11 ] ACSF [1-11], Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asn-Lys-Gly-Lys-Ser-Ile-Gln ([Asn.sup.10 ]ACSF [1-16]) and [Asn.sup.10,Tyr.sup.17 ] ACSF (1-17). The analogs, [Glu.sup.8,Asn.sup.10,Cys.sup.11 ] ACSF (1-11) and [Asn.sup.10 ]ACSF (1-16) were inactive in the adenylate cyclase assay and did not antagonize the action of PTH itself or of conditioned medium from BEN cells. [Glu.sup.8,Asn.sup.10,Cys.sup.11 ] ACSF (1-11) conjugated to soya bean trypsin inhibitor was used to immunize rabbits against ACSF, and an antiserum produced which was used in radioimunoassay.
In order to treat patients afflicted with HHM it is necessary to provide an ACSF antagonist. It is a first objective herein to obtain DNA encoding ACSF in order to obtain the complete amino acid sequence thereof. This will facilitate the preparation of ACSF and ACSF antagonists in recombinant cell culture. In addition, C-terminal sequence for ACSF will allow one to prepare antibodies specific for this domain of ACSF, thereby improving immunoassays for ACSF. These and other objects of the invention will be apparent from consideration of the specification in its entirety.